The principal goal of this grant proposal is to obtain information about the control of transcription of mitochondrial DNA. The purpose is to gain better insight to the mechanisms that control mitochondrial biogenesis in both the lower and higher eukaryotes. The work is particularly relevant to cardiac tissue in which metabolism is almost completely dependent upon the aerobic synthesis of ATP generated by mitochondria. Our specific aims are to characterize the transcriptional apparatus of the yeast mitochondria, which serve as an important model of mammalian mitochondria. In particular we aim to purify the mitochondrial RNA polymerase, to characterize its interaction with mitochondrial promoter and other mitochondrial DNA sequences. We plan to isolate the genes of the polymerase subunits, with special emphasis on a 70 kd peptide transcription factor. In vivo mutagenesis of these subunit genes will be undertaken. The generation of mutants in these genes will enable use to undertake structure function studies and to isolate other factors involved in mitochondrial transcription. The biogenesis and import into the mitochondria of the RNA polymerase will be studied intensively both in vivo and in vitro using a new procedure recently developed in our laboratory for the measurement of transcripts. Attenuation of mitochondrial transcription has recently been discovered in our laboratory and we will develop the tools to study the site and possible mechanism of transcriptional attenuation.